Protransduzin b, a gene transfer enhancer

ABSTRACT

An N-terminally protected peptide having the sequence 
                 (SEQ ID NO: 1)     X-Glu-Cys-Lys-Ile-Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln,                
wherein X is a group protecting the N-terminal of the peptide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of application Ser. No.14/787,160, filed Oct. 26, 2015, which claims priority to a NationalStage filing of PCT Application No. PCT/EP2014/058870, filed Apr. 30,2014, which claims priority to European Application No.: 13166266.0,filed May 2, 2013, all of which are incorporated herein by reference.

TECHNICAL FIELD

The present application relates to an N-terminally protected peptide, toa medicament containing said peptide, to said peptide for use in genetherapy, to a method for enhancing the infection of a cell by agenetically engineered viral construct, and to the use of said peptidefor amplification for transfection or transduction.

BACKGROUND

The importance of genetic engineering has increased in recent yearsbecause of an enormous progress in the applied methods, because it ispredictable that not only the production of protein/peptide activesubstances, but also the transfection of cells with stable genes as alaboratory tool and ultimately the introduction of genes in cells as aremedy for gene defects will be highly relevant to the therapy ofnumerous diseases.

SUMMARY AND INTRODUCTION

The introduction of genetic material for changing specific cellfunctions has become an indispensable tool of biological-medical basicand applied research since the cloning of the first human genes andrecombinant production, since the methods of gene transfer undergocontinuous progress with increasing efficiency. Numerous methods of geneintroduction have led to optimization. The corresponding experienceshave been collected over many years of history, which was very slow atfirst.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a high pressure liquid chromatography (HPLC) chromatogram ofprotransduzin A and protransduzin B.

DETAILED DESCRIPTION

Even before the elucidation of the function of deoxyribonucleic acid(DNA) in 1953 by F. Crick and J. Watson, F. Griffith had succeededtowards the end of the 1920's in experiments in transforming apathogenicPneumococcus strains into pathogens. This transformation was due to alucky circumstance, because the pneumococci had a rare naturalcompetence of DNA uptake. A specific introduction of DNA intoprokaryotes was achieved by J. Lederberg, M. Delbrück and S. Luria,among others, by means of phages, the so-called transduction. With theestablishing of cell culture, the culturing of eukaryotic cells under invitro conditions, a number of physical and chemical methods fortransfection have been developed. The physical methods, which are morefrequently utilized, but require more expensive equipment, includeelectroporation and microinjection, which competed with the more simplyapplicable chemical methods, such as the calcium phosphate precipitationmethod usual in the 1980's and still today, or the methods widespread inthe early 1990's, which were based on cationic lipids or cationicpolymers. However, the use of these methods has always been dependent onthe cells or the DNA. Also, the DNA introduced into the cells wasgenerally extrachromosomal (transient transfection), and thus it was notpassed on to the daughter cells. However, phages (e.g., lambda phage)were known to be able to integrate their DNA into the host genome(prophage, lysogenic infection pathway). From here, it was only a smallstep (1981/1982) to the “Establishing of retroviruses as gene vectors”(by Doehmer et al. and Tabin et al.). Viruses are species-specific andorgan/tissue-specific, which is why all viruses do not infect all(eukaryotic) cells. Alterations in the viral envelope (exchange ofglycoproteins, so-called pseudotyped viruses) and additions of mostlycationic peptides are supposed to enhance transduction efficiency.

First enhancers of the uptake of virus particles attracted attention inthe study of HIV. During analyses of in vitro infection by means of aspecific cell test, the inhibition of the fusion of HIV by bloodfiltrate peptides was observed (Munch et al., VIRIP).

It has been shown that these fragments of proteins, which surprisinglyare naturally occurring, form fibrous structures as enhancers in humansperm, “Semen derived Enhancer of Virus Infection” (SEVI), which arecharacterized as amyloid fibrils. These nanofibrils enhance the dockingof viruses to their target cells, increasing the rate of viral infectionby several powers of ten.

This was utilized for improving retroviral gene transfer for basicresearch and for possible future therapeutic applications. Thus, itcould be shown that lentiviral and gamma-retroviral viruses, which areused for gene therapy, exhibit a many times higher gene transfer rate inthe presence of the SEVI protein for different cell types, such as humanT cells, cervical carcinoma cells, leukemia cells, hematopoietic stemcells, and embryonic stem cells (Wurm et al., J. Gene Med. 2010, 12,137-46; Wurm et al., Biol. Chem. 2011, 392, 887-95).

Studies for the development of further enhancers, such as SEVI andseminogelin, led to the assumption that peptides from viral envelopeproteins may also be suitable as enhancers of transfection, whichsurprisingly was an unexpectedly great success (Maral Yolamanova, NatureNanotechnology). Thus, it could be shown, for example, that HIVspreincubated with different concentrations (1-100 μg/ml) ofprotransduzin A (synonym: EF-C) exhibit an infection rate with reportercells that is increased by several powers of ten. As the mechanism ofaction, it was assumed that EF-C forms fibrillary structures that arecapable of binding and concentrating viruses and accordingly amplifyingthe entry of the viruses into the cell. In addition to the infectionwith viral particles, EF-C enhances the transduction of lentiviral andretroviral particles with high efficiency in a wide variety of humancell types (T cells, glial cells, fibroblasts, hematopoietic stem cells)applied in gene therapy (Jan Munch et al., Nature Nanotechnology, Vol.8, No. 2, pp. 130-136). EP 2 452 947 A1 also relates to protransduzin A.Because of the increasing importance of gene technology as set forthabove, more effective enhancers of gene transfer are desirable. Theobject of the invention is to provide an improved enhancer of genetransfer.

Surprisingly, it has been found that an N-terminally protected peptidehaving the sequence

(SEQ ID NO: 1) X-Glu-Cys-Lys-Ile-Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln,wherein X is a group protecting the N-terminal of the peptide, achievesthe object of the invention. In particular, X represents one or twoalkyl groups, such as methyl, ethyl, propyl or butyl groups, an acylgroup, such as an acetyl or propionyl group, or the group X-Glu is theamino acid pyroglutamic acid:

Surprisingly, it has been found that it is the modification of theN-terminal end by pyroglutamic acid in vitro (without cellularinfluences, especially the presence of enzymes), in particular, thatresults in an enormous increase in stability of the protransduzin inaqueous solution. This is clear from the results shown in FIG. 1.

In the left column of FIG. 1 (HPLC chromatogram), results forprotransduzin A upon storage for 0-13 days at −20° C. and at 4° C. (13days) are compared with the results for protransduzin B under the sameconditions. It is clear that protransduzin A is degraded almost to onehalf upon storage at 4° C. for 13 days, whereas protransduzin B ishardly degraded at all under the same storage conditions (the height ofthe peaks corresponds to the concentration of the components containedin the sample).

In Journal of Biological Chemistry, Vol. 286, No. 45, pp. 38825-38832,S. Jawhar et al. report on the state of the science relating to amyloidpeptides, especially pyroglutamate-modified amyloid polypeptides. Suchamyloid polypeptides have a large number of amino acids and arebasically not comparable to short-chained peptides, to which thoseaccording to the invention also belong. Incidentally, this mini reviewrelates to cellular events occurring under in vivo conditions in thepresence of enzymes, which is by no means comparable, however, to theconditions under which the stability of protransduzin has been improvedaccording to the invention, i.e., in vitro conditions.

The peptide according to the invention may also be used as a medicament.

The invention also relates to the use of the peptide according to theinvention in gene therapy for treating diseases that are treatable withgene therapy.

The invention also relates to a method for enhancing the infection of acell by a virus, comprising the steps:

-   -   providing the peptide according to claim 1 dissolved in an        organic solvent;    -   adding the peptide to an aqueous solution to form insoluble        aggregates of the peptide;    -   mixing the solution from the last preceding step; and    -   culturing the cells.

The present invention also relates to the use of the peptide accordingto the invention for enhancing the infection of a cell with a virus.

Finally, a kit containing the peptide according to the invention is alsoclaimed.

The peptide according to the invention (protransduzin B) can beprepared, for example, by the method according to Merrifield withFmoc-protected amino acids.

This method works with Fmoc-protected derivatives, i.e., with(9-fluorenylmethoxycarbonyl)-protected amino acids, in a stepwise solidphase synthesis according to the Merrifield principle, especially on aWang resin preloaded with Fmoc-L-glutamine (0.59 mmol/g, 100-200 mesh)as a solid support on the synthesizer ABI-433.

The activation of the Fmoc-L-amino acids, which were typically employedin a tenfold molar excess, is performed with[(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate] (HBTU, 100 mmol/l) with additions of 0.5 M1-hydroxybenzotriazole (HOBt) and 2 M diisopropylethylamine (DIEA) inN-methyl-2-pyrrolidinone (NMP) at room temperature.

The individual acylation reactions take 45 minutes, and the Fmocdeprotection with 20% piperidine takes 15 minutes.

The following amino acid derivatives and related orthogonalacid-cleavable side chain protective groups are employed for synthesis:

Fmoc-L-Asn(Trt), Fmoc-L-Cys(Trt), L-pGlu, Fmoc-L-Gln(Trt), Fmoc-L-Ile,Fmoc-L-Lys(Boc), Fmoc-L-Met and Fmoc-L-Trp(Boc).

After cleaving the resin support from the peptidyl resin with 94%trifluoroacetic acid (TFA), 3% ethanedithiol (EDT) and 3% demineralizedwater, the raw peptide is precipitated in cold tert-butyl methyl ether,the raw peptide is centrifuged off as a pellet, and the supernatant isdiscarded.

The subsequent chromatographic purification of the raw peptide iseffected in a preparative way by gradient elution.

The difference between protransduzin A according to EP 2 452 947 A1 andprotransduzin B resides in the fact that the synthetic L-pyroglutamicacid (pGlu) is inserted N-terminally in exchange for syntheticL-glutamine (Gln) in protransduzin B. The original glutamine is modifiedby ring closure to form a lactam.

Purification:

Preparative separation: The purification is performed on an HPLC fromthe Gilson Company. The UV/VIS detector is from the Kronwald Company,and the separation is detected at the wavelength 230 nm. The flow rateis 40 ml/min.

The column is a Waters Prep-Pak C18 cartridge (47×300 mm).

Eluent A: 0.1% TFA in demineralized water; eluent B: 0.1% TFA in 80%acetonitrile and 20% demineralized water.

The gradient for protransduzin B is 35%-55% eluent B in 40 min, i.e.,0.5% eluent B per minute. Protransduzin B elutes at 40% eluent B and iscollected in several fractions of 0.5 to 1 min. The analytically cleanfractions are pooled and lyophilized.

Processing for Application: Lyophilization:

The unit Epsilon 1/45 of the Christ company, whose technical data areset as follows, is used for freeze drying: shelf area 3.78 m²; icecapacity about 60 kg; ice condenser performance max. 45 kg/24 h; finalpartial pressure of vacuum pump 1×10⁻³/10⁻⁴ mbar with/without gasballast; freeze-drying data (unit operated manually with gas ballast):final partial pressure 1×10⁻² mbar; ice condenser temperature −50° C.;shelf temperature+15° C.; operating point of shelf heating 0.5 mbar;freeze-drying time up to 3 days.

Transduction of Cells with Protransduzin B

Dissolve 0.5 mg of protransduzin B in 50 μl of DMSO. Then add 450 μl ofPBS to the solution, fibrils forming within 3 min. Add this stocksolution (1 mg/ml) to the vectors to obtain a concentration ofprotransduzin B of 25 μg/ml. Vortex the solution for 1 min, thencentrifuge with 5000 g for 5 min. The supernatant is discarded, and thepellet is suspended in a little PBS and added to the cells. The cellsare incubated in an incubator for 2 days.

The transduction rate is significantly enhanced by protransduzin B. Upto 96% of the cells can be transduced by means of protransduzin B.

1. An N-terminally protected peptide having the sequenceX-Glu-Cys-Lys-Ile-Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln, wherein X is a groupprotecting the N-terminal of the peptide.
 2. The peptide according toclaim 1, wherein X is one or two alkyl groups, such as methyl, ethyl,propyl or butyl groups, an acyl group, such as an acetyl or propionylgroup, or the group X-Glu is the amino acid pyroglutamic acid.
 3. Amedicament containing a peptide according to claim
 1. 4. The peptideaccording to claim 1 for use in gene therapy for treating diseases thatare treatable with gene therapy.
 5. A method for enhancing the infectionof a cell by a virus, comprising the steps: providing the peptideaccording to claim 1 dissolved in an organic solvent; adding the peptideto an aqueous solution to form insoluble aggregates of the peptide;mixing the solution from the last preceding step; and culturing thecells.
 6. A method for enhancing the infection of a cell by a viruscomprising administering to a patient a peptide according to claim
 1. 7.A kit containing a peptide according to claim 1.